The present invention concerns fibroblast growth factor 9 (FGF9), a novel high affinity ligand for fibroblast growth factor receptor 3 (FGFR3), methods for detecting FGFR3 using said ligand and pharmaceutical compositions for modulating FGFR3 activity comprising FGF9, an antagonist thereof, or FGF9 binding-agents.
Fibroblast growth factors (FGF) comprise a family of at least nine multifunctional polypeptides involved in a variety of biological processes including morphogenesis, angiogenesis and tissue remodeling. They stimulate the proliferation of cells from mesenchymal to epithelial and neuroectodermal origin. FGFs share structural similarity, but differ in their target specificity and spatial and temporal expression pattern. Four FGF receptor (FGFR) genes encoding transmembrane protein tyrosine kinases, have been cloned and identified in mammals and their homologues described in birds, Xenopus and Drosophila (Givol and Yayon, FASEB J., 6:33622269 (1992)). The actual number of functional receptor proteins is however much greater since multiple variants are generated, as cell bound or secreted forms, by alternative RNA splicing and multiple polyadenylation sites. Beside these high affinity receptors, FGFs bind tightly to low affinity, high capacity binding sites identified as heparan sulfate proteoglycans (HSPGs). These heparan sulfates modulate FGF-receptor binding and biological activity and serve as an obligatory integral component in the formation of a functional tertiary complex between FGF, FGFR and the appropriate HSPG.
In light of the large number of ligand and receptor variants, a major question regarding FGF function is their ligand-receptor specificity. Both FGFR1 and FGFR2 bind acidic FGF/FGF1 and basic FGF/FGF2 with similar affinity (Dionne et al., EMBO J., 9:2685-2692 (1990)). In fact all FGFRs tested so far bind FGF1 and FGF4 (hst/kfgf) with moderate to high affinity, demonstrating an apparent redundancy in the FGF system. In contrast to FGFRs 1 and 2, FGFR3 was found to bind only FGF1 and FGF4 albeit with moderate affinity (Ornitz and Leder, J. Biol. Chem., 267:16305-16311 (1992); Chellaiah et al., J. Biol. Chem., 269(15):11620-11622, (1994)). No specific ligand has been identified so far, for either of the spliced forms of this receptor.
Recently, mutations in FGFR3 have been shown to be responsible for achondroplasia, the most common form of genetic dwarfism. Examination of the sequence of FGFR3 in achondroplasia patients identified a mutation in the transmembrane domain of the receptor.
The focus of FGFR3 as the receptor involved in achondroplasia raised the need for a specific ligand for this receptor, which does not substantially bind to the other three FGFRs, both for the purpose of research and study of this disease as well as for the purpose of developing possible medicaments for its treatment.
A heparin-binding, glia-activating factor purified from the culture supernatant of a human glioma cell-line was found, by a homology search, to be the ninth member of the FGF family and was thus termed FGF9. Human FGF9 was found to code for a 208 amino acid protein and presents a unique spectrum of biological activity as it stimulates the proliferation of glial cells, PC-12 cells and BALB/C 3T3 fibroblasts, but nevertheless is not mitogenic for endothelial cells (Miyamote et al., Mol. Cell. Biol., 13(7):4251-4259 (1993); Naro et al., J. Biol. Chem., 267:16305-16311 (1993)).
The present invention is based on the surprising finding their fibroblast growth factor 9 (FGF9) is a high affinity (kD: 0.25 nM) ligand for fibroblast growth factor receptor 3 (FGFR3) which does not bind to FGFR1 or FGFR4 and binds to FGFR2 only at a substantially lower affinity.
Thus, the present invention provides for the first time a specific ligand for FGFR3 being a fibroblast growth factor 9 (FGF9). This specific FGFR3 ligand may be used both for detection and for therapeutical treatment purposes.
This specific novel ligand for FGFR3 may be used in a method for the detection of FGFR3 in a sample or tissue comprising:
(i) contacting the sample or tissue with FGF9 and allowing formation of receptor-ligand pairs, and
(ii) detecting the presence of FGFR3-FGF9 pairs, a positive detection indicating the presence of FGFR3 in the sample or tissue.
The sample may be a sample of body fluid such as blood, in which soluble FGFR3 is present and the tissue may be a tissue obtained from a patient, for example by cartilage biopsy or alternatively, may be a tissue within the body of an individual and in such a case the detection is carried out in vivo.
Detection may be carried out for example by labelling the FGF9 with a suitable detectable label, and then determining whether any label is bound to proteins in the sample or to the surface of cells in the tissue which is assayed for the presence of FGFR3. Alternatively detection may be carried out by using labeled antibodies against FGF9, capable of recognizing FGF9 which is bound of FGFR3.
In accordance with the present invention, it was found that FGF9 is a heparin-dependent ligand for FGFR3. Thus, in accordance with the method of detection of FGFR3 by use of the FGF9 ligand, it is preferable that heparin would also be present in the detection medium.
In accordance with the present invention, it was further found that FGF9 not only specifically binds to the FGFR3, but also specifically activates this receptor without activating the FGFR1 and FGFR4 receptors and, if appropriate concentrations are chosen, without significantly activating FGFR2. This finding leads to the preparation of pharmaceutical compositions comprising a pharmaceutically acceptable carrier and as an active ingredient a therapeutically effective amount of FGF9. Such a pharmaceutical compositions may be used for stimulating the activity of FGFR3.
This finding also leads to the preparation of pharmaceutical compositions comprising a pharmaceutically acceptable carrier and as an active ingredient an antagonist of the FGF9, or an FGF9 binding agent an example being an antibody against FGF9.
Pharmaceutical compositions comprising an antagonist of FGF9 may be used to attenuate directly the activity of the FGFR3, and pharmaceutical compositions comprising an FGF9 binding agent such as an antibody against FGF9, may neutralize circulating native FGF9 and thus attenuate indirectly the activity of FGFR3.
Normal cartilage and bone growth and repair of damage to the cartilage and bone requires a specific and delicate balance between up regulation and down regulation of the activity of the FGFR3. Without wishing to be bound by theory, it is assumed that active FGFR3 is necessary in the initial stages of cartilage-bone differentiation, and after differentiation is required for cartilage-bone repair. Thus, the pharmaceutical composition comprising as an active ingredient FGF9, which stimulates the activity of FGFR3, may be used in order to encourage cartilage and bone repair, for example by administration to the site of injury. Furthermore, FGFR3 exists usually temporarily on mesenchymal stem cells and usually disappears after differentiation. Administration of FGF9 may serve to stabilize FGFR3 and thus prolong the period in which it is active prior to differentiation. FGF9 has also a chemotactic affect of FGFR3-carrying cells and can promote migration of such FGFR3 carrying cells, typically mesenchymal stem cells, to a desired site, for example, by injection of FGF9 to the growth plate top of the column.
According to this theory, overactivation of FGFR3 after the stage of initial differentiation of bone and cartilage cells, leads to halted growth, and is probably the cause of achondroplasia. Thus, a pharmaceutical composition comprising as an active ingredient an antagonist of FGF9 which attenuates the activity of FGFR3, or comprising an FGF9 binding agent (such as an antibody against FGF9), which neutralizes native circulating FGF9, should be used in cases of overactivity of the FGFR3 receptor in differentiated tissues, which causes bone and cartilage growth arrest. Such bone and cartilage growth arrest may lead to achondroplasia dwarfism, or other abnormalities of bone and cartilage growth, for example, multiple hereditary exostosis, solitary hereditary exostosis, hallux valgus deformity, synovial chondromatosis and endochondromas.
The above conditions may be treated with a pharmaceutical composition comprising either an antagonist of FGF9, or an FGF9 binding agents capable of neutralizing native circulating FGF9, which both serve to attenuate the activity of the FGFR3.
The present invention also concerns a novel recombinant mouse FGF9, and a novel recombinant chicken FGF9, as well as DNA sequences coding for these novel recombinant proteins.
The present invention still further concerns an expression vector comprising the sequence of FGF9 under the expression control of a strong promoter such as the CMV or SV40 or a cartilage/bone promoter such as collagen type-2 promoter. Such an expression vector may be used to produce a transgenic mammal, which over-expresses FGF9 leading to overactivation of the FGFR3 receptor and thus to halted growth. Such an animal may serve as a model for diseases and disorders resulting from halted growth, such as genetic achondroplasia.